Anti-emap ii antibody and use thereof

ABSTRACT

The present invention relates to an anti-EMAP II antibody or an antigen-binding fragment thereof, a nucleic acid molecule for coding the same, or a preparation method thereof. Also, the present invention provides a method for using the anti-EMAP II antibody or the antigen-binding fragment thereof in preventing, treating or diagnosing EMAP II-mediated diseases, for example, TNF-α-mediated disease or Alzheimer&#39;s disease. Furthermore, the present invention provides a method for using the anti-EMAP II antibody or the antigen-binding fragment thereof in detecting or quantifying an EMAP II antigen. The antibody of the present invention may exhibit a more improved antigen-binding capacity compared to existing anti-EMAP antibodies and may treat the TNF-α-mediated disease more effectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims priority to U.S.application Ser. No. 16/497,612, having a 371 completion date of Sep.25, 2019, which is a U.S. National Stage application, and claimspriority of International Application No. PCT/KR2018/003569, filed Mar.27, 2018, which claims priority of South Korean Application No.10-2017-0038706, filed March 27, 2017. The contents of all of the priorapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an anti-EMAP II antibody or anantigen-binding fragment thereof, a nucleic acid molecule for coding thesame, or a preparation method thereof.

The present invention also relates to a method for using the anti-EMAPII antibody or the antigen-binding fragment thereof in preventing,treating or diagnosing EMAP II-mediated diseases, for example,TNF-α-mediated disease or Alzheimer's disease.

Furthermore, the present invention relates to a method for using theanti-EMAP II antibody or the antigen-binding fragment thereof indetecting or quantifying an EMAP II antigen.

BACKGROUND

An endothelial monocyte activating polypeptide II (EMAP II) is producedin such a way that a C-terminal region of p43 protein, i.e., a precursorprotein, is isolated by means of an activated caspase-7 while apoptosisprogresses (Behrensdorf et al., FEBS Lett. 466:143-147, 2000).

EMAP II, which is a factor for mediating pro-inflammatory responses,induces a tissue factor, a tumor necrosis factor (TNF) and interleukin-8(IL-8) to be expressed in mononuclear phagocytes and polymorphonuclearleucocytes. Macrophages are accumulated in a tissue where EMAP II mRNAis overexpressed. This means that EMAP II is a chemotactic substancewhich induces the macrophages to dead cells. It is known that EMAP IIacts as a cytokine and 15 amino acids of EMAP II N-terminus play animportant role (Knies, U. E. et al., PNAS USA, 95:12322-12327, 1998,etc.).

The Korean Patent Publication No. 2010-0093451 discloses an anti-EMAP IImonoclonal antibody specific to EMAP II (also referred to as “SCYE1”)protein, and shows that such antibody may be used in diagnosing andtreating inflammatory diseases; inhibits secretion of TNF-α mediatinginflammatory responses; and exhibits an effect on treating Alzheimer'sdisease. Also, the Korean Patent Publication No. 2012-0118918 disclosesan anti-EMAP II chimera or humanized antibody, of which immune responsesare minimized in the human body, and shows that such antibody has thesame binding capacity as a parent monoclonal antibody specificallybinding to EMAP II.

If there is an antibody variant having an addition, deletion orsubstitution of an amino acid at a certain site in a heavy chain and/orlight chain variable region of a parent antibody, and if such antibodyvariant shows a more improved antigen-antibody binding capacity than theparent antibody, it is found that such antibody variant may beadministered more stably with less side effects, while exhibiting atherapeutic effect equal to or more than that of the parent antibodyeven in a smaller dosage thereof.

Against such technological backdrops, the present inventors have madeevery endeavor to develop an antibody specifically binding to EMAP IIwith a more improved antigen-binding capacity, and thus have developed anovel antibody with a more improved EMAP II binding capacity and a moreexcellent effect of inhibiting TNF-α and treating related diseases thanexisting antibodies, thereby completing the present invention.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

One objective of the present invention is to provide an anti-EMAP IIantibody or an antigen-binding fragment thereof with a more improvedEMAP II antigen-binding capacity.

Other objective of the present invention is to provide a nucleic acidmolecule for coding the antibody or the antigen-binding fragmentthereof; a recombinant vector containing the nucleic acid molecule; ahost cell having the recombinant vector transferred thereinto; and amethod for producing the anti-EMAP II antibody or the antigen-bindingfragment thereof by using the host cell.

Another objective of the present invention is to provide a compositionfor detecting an EMAP II antigen, containing the antibody or theantigen-binding fragment thereof; a kit for detection containing thesame; and a method for detecting the EMAP II antigen by using thecomposition and the kit.

Yet another objective of the present invention is to provide acomposition for preventing or treating EMAP II-mediated diseases, forexample, TNF-α-mediated disease or Alzheimer's disease, containing theantibody or the antigen-binding fragment thereof.

Technical Solution

This is described in detail as follows. Meanwhile, each description andembodiment disclosed in the present invention may be applied to otherdescriptions and embodiments thereof, respectively. In other words, allthe combinations of various elements disclosed in the present inventionfall within the scope of the present invention. Also, it may not be seenthat the scope of the present invention is limited to the specificdescriptions described below.

1. Anti-EMAP II Antibody and Antigen-Binding Fragment Thereof

In one aspect of the present invention for achieving the objectives,there is provided an anti-EMAP II antibody or an antigen-bindingfragment thereof with an improved antigen-binding capacity.

The anti-EMAP II antibody or the antigen-binding fragment thereofaccording to the present invention binds to EMAP II with a higheraffinity compared to existing antibodies and inhibits an activitythereof, thereby exhibiting an excellent TNF-α inhibition effect. Thus,such antibody may be valuably used in preventing or treating EMAPII-mediated diseases, for example, TNF-α-mediated disease or Alzheimer'sdisease for itself or along with conventional, pharmaceuticallyacceptable carriers, etc.

The anti-EMAP II antibody of the present invention contains a lightchain variable region, including a light chain CDR1 consisting of SEQ IDNO: 7, a light chain CDR2 consisting of SEQ ID NO: 9, and a light chainCDR3 consisting of SEQ ID NO: 11; and a heavy chain variable region,including a heavy chain CDR1 consisting of SEQ ID NO: 12, a heavy chainCDR2 consisting of SEQ ID NO: 14, and a heavy chain CDR3 consisting ofSEQ ID NO: 16.

In one specific embodiment of the anti-EMAP II antibody according to thepresent invention, the anti-EMAP II antibody may contain a light chainvariable region consisting of SEQ ID NO: 3; and a heavy chain variableregion consisting of SEQ ID NO: 4.

Preferably, the antibody may be a humanized antibody, and particularlymay contain a human kappa or IgG-derived constant region, but is notlimited thereto.

As used herein, the term “antibody” means a protein molecule serving asa receptor, which specifically recognizes an antigen, including animmunoglobulin molecule immunologically having reactivity with a certainantigen. As an example, such antibody may include a monoclonal antibody,a polyclonal antibody, a full-length antibody and an antibody fragmentall. Also, the term “antibody” may include a bivalent ordual-specificity molecule (e.g., a dual-specificity antibody), adiabody, a triabody or a tetrabody.

As used herein, the term “monoclonal antibody” refers to an antibodymolecule of a single molecular composition obtained from a group ofsubstantially identical antibodies, wherein this monoclonal antibodyshows a single binding property and affinity to a certain epitope, whilethe polyclonal antibody may bind to several epitopes. As used herein,the term “full-length antibody” takes on a structure of having twofull-length light chains and two full-length heavy chains, wherein eachlight chain is linked to a heavy chain by disulfide bonding. A heavychain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ) andepsilon (ε) type, and also has a gamma 1 (γ1), gamma 2 (γ2), gamma 3(γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) as a subclass. A lightchain constant region has a kappa (κ) and lambda (λ) type. IgG includesIgG1, IgG2, IgG3 and IgG4 as a subtype.

As used herein, the term “heavy chain” may include both a full-lengthheavy chain and a fragment thereof, containing a variable region VHincluding an amino acid sequence with a variable region sequence enoughto give specificity to an antigen; as well as three constant regionsCH1, CH2 and CH3. Also, as used herein, the term “light chain” mayinclude both a full-length light chain and a fragment thereof,containing a variable region VL including an amino acid sequence with avariable region sequence enough to give specificity to an antigen; aswell as a constant region CL.

As used herein, the term “antibody variant” means a variant, in which apart of an amino acid sequence of a parent antibody is substituted,added or deleted to improve an antibody affinity, etc., more than theparent antibody, and includes a chimera antibody, a humanized antibody,etc. The antibody of the present invention, which is a variant of anantibody disclosed in the Korean Patent Publication No. 2012-0118918, isused herein along with an “anti-EMAP II antibody” and an “anti-EMAP IIantibody variant.”

As used herein, the term “chimera antibody,” which is created byrecombining a variable region of a mouse antibody and a constant regionof a human antibody, is an antibody which has a great improvement inimmune responses compared to the mouse antibody.

As used herein, the term “humanized antibody” means an antibody, whichis modified in such a way that a protein sequence of an antibody derivedfrom non-human species becomes similar to an antibody naturally producedfrom humans. As an example, the humanized antibody may be prepared byrecombining a mouse-derived CDR with a human antibody-derived FR toprepare a humanized variable region, and then by recombining the samewith a constant region of a preferable human antibody. However, if onlyCDR grafting is simply performed, the affinity of the humanized antibodybecomes low. Thus, such low affinity may be raised up to the same levelas the affinity of an original mouse antibody, in such a way thatseveral important FR amino acid residues considered to have an influenceon a three-dimensional structure of the CDR are allowed to have moreaffinity with those of the mouse antibody.

The present invention also provides an antigen-binding fragment of theanti-EMAP II antibody. The antibody-binding fragment may be Fab, F(ab′),F(ab′)2 or Fv.

As used herein, the terms “fragment,” “antibody fragment” and“antigen-binding fragment” refer to any fragments of the inventiveantibody, which hold an antigen-binding function of the antibody,wherein such terms are interchangeably used with each other. Anexemplary antigen-binding fragment includes Fab, Fab′, F(ab′)2, Fv andthe like, but is not limited thereto.

The Fab has one antigen-binding site, which takes on a structure ofhaving light chain and heavy chain variable regions; a light chainconstant region; and a first constant region (CH1 domain) of a heavychain. With regard to an antibody molecule, its antigen-binding fragmentor an antibody fragment means a fragment which holds an antigen-bindingfunction, wherein Fab′ has a difference from Fab, in that Fab′ has ahinge region containing at least one cysteine residue at C terminus of aheavy chain CH1 domain. F(ab′)2 antibody is produced in such a way thata cysteine residue of the hinge region of Fab′ forms disulfide bonding.Fv is a minimum antibody fragment having only a heavy chain variableregion and a light chain variable region, wherein a recombinationtechnology for producing an Fv fragment is disclosed in theInternational Patent Publication filed under the patent cooperationtreaty (PCT) WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, WO88/09344 and the like. In case of two-chain Fv, a heavy chain variableregion and a light chain variable region are linked to each other bymeans of non-covalent bonding. In case of single-chain Fv, a heavy chainvariable region and a single chain variable region are linked to eachother by means of covalent bonding generally via a peptide linker, ordirectly linked to each other at C-terminus, and thus may form astructure like a dimer, as shown in the two-chain Fv. Such antibodyfragment may be obtained by using protease (for example, Fab may beobtained by performing restriction digestion of a whole antibody withpapain, while F(ab′)2 fragment may be obtained by doing so with pepsin),and may be preferably prepared by means of a gene recombinationtechnology.

Also, the antibody or the antigen-binding fragment thereof according tothe present invention may contain a sequence of the anti-EMAP IIantibody described herein as well as biological equivalents thereof,within the range that may show an improvement in the EMAP IIantigen-binding capacity. For example, an amino acid sequence of theantibody may be further given a change, in order to more improve thebinding affinity and/or other biological characteristics of theantibody. Such modification includes, for example, the deletion,insertion and/or substitution of an amino acid sequence residue of theantibody. Such amino acid mutation is performed on the basis of therelative similarity of amino acid side chain substituents, e.g.,hydrophobicity, hydrophilicity, charges, sizes, etc. According to ananalysis of sizes, shapes and types of the amino acid side chainsubstituents, it might be seen that arginine, lysine and histidine areall positively charged residues; alanine, glycine and serine havesimilar sizes; and phenylalanine, tryptophan and tyrosine have similarshapes. Thus, in this regard, it might be seen that arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine are biologically functional equivalents.

In one specific embodiment of the present invention, the EMAP IIantibody of the present invention has an amino acid sequence with onlyfour amino acids substituted therein compared to another antibody, i.e.,a comparison target, but shows such a remarkable effect that anantigen-antibody binding capacity of the former one is improved 10 timesmore than the latter one (FIGS. 2 and 3).

Also, in another specific embodiment of the present invention, it wasidentified in case of administering the inventive EMAP II antibody intoan arthritis-induced mouse that such case achieves an excellent effectof inhibiting TNF-α secretion as well as an excellent effect of treatingarthritis symptoms (FIGS. 4 and 5).

2. Nucleic Acid Molecule for Coding EMAP II Antibody or Antigen-BindingFragment Thereof; Recombinant Vector; Transformant; and Method forPreparing the Same 2-1. Nucleic Acid Molecule for Coding EMAP IIAntibody or Antigen-Binding Fragment Thereof

In another aspect of the present invention, there is provided a nucleicacid molecule for coding the antibody or the antigen-binding fragmentthereof. The antibody and the antigen-binding fragment thereof are thesame as described above.

As used herein, the term “nucleic acid molecule” has a meaningcomprehensively including DNA (gDNA and cDNA) and RNA molecules, whereina nucleotide, i.e., a basic unit of the nucleic acid molecule, includesa natural nucleotide as well as an analogue with a sugar or base sitemodified therein (Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman and Peyman, Chemical Reviews (1990) 90:543-584). Asequence of the nucleic acid molecule for coding light chain and heavychain variable regions according to the present invention may bemodified, wherein the modification includes the addition, deletion, ornon-conservative or conservative substitution of the nucleotide.

According to one exemplary embodiment of the present invention, thenucleic acid molecule for coding an antibody or an antigen-bindingfragment thereof according to the present invention may include anucleic acid molecule for coding a light chain CDR1 consisting of aminoacid sequence of SEQ ID NO: 7, a nucleic acid molecule for coding alight chain CDR2 consisting of amino acid sequence of SEQ ID NO: 9, anda nucleic acid molecule for coding a light chain CDR3 consisting ofamino acid sequence of SEQ ID NO: 11; and a nucleic acid molecule forcoding a heavy chain CDR1 consisting of amino acid sequence of SEQ IDNO: 12, a nucleic acid molecule for coding a heavy chain CDR2 consistingof amino acid sequence of SEQ ID NO: 14, and a nucleic acid molecule forcoding a heavy chain CDR3 consisting of amino acid sequence of SEQ IDNO: 16.

In a preferred aspect of the exemplary embodiment above, the nucleicacid molecule may include a nucleic acid molecule for coding a lightchain variable region consisting of amino acid sequence of SEQ ID NO: 3,and a nucleic acid molecule for coding a heavy chain variable regionconsisting of amino acid sequence of SEQ ID NO: 4, but is not limitedthereto.

2-2. Recombinant Vector Containing Nucleic Acid Molecule

In another aspect of the present invention, there is provided arecombinant vector containing the nucleic acid molecule.

As used herein, the term “vector,” which serves as a means forexpressing a target gene in a host cell, includes a plasmid vector; acosmid vector; a virus vector such as a bacteriophage vector, anadenovirus vector, a retrovirus vector and an adeno-associated virusvector; and the like, and may be preferably the plasmid vector, but isnot limited thereto.

According to one exemplary embodiment of the present invention, therecombinant vector containing the nucleic acid molecule according to thepresent invention may contain a base sequence of a light chain variableregion, including a nucleic acid molecule for coding a light chain CDR1consisting of amino acid sequence of SEQ ID NO: 7, a nucleic acidmolecule for coding a light chain CDR2 consisting of amino acid sequenceof SEQ ID NO: 9, and a nucleic acid molecule for coding a light chainCDR3 consisting of amino acid sequence of SEQ ID NO: 11; and/or a basesequence of a heavy chain variable region, including a nucleic acidmolecule for coding a heavy chain CDR1 consisting of amino acid sequenceof SEQ ID NO: 12, a nucleic acid molecule for coding a heavy chain CDR2consisting of amino acid sequence of SEQ ID NO: 14, and a nucleic acidmolecule for coding a heavy chain CDR3 consisting of amino acid sequenceof SEQ ID NO: 16.

In a preferred aspect of the exemplary embodiment above, the vector maycontain a nucleic acid molecule for coding a light chain variable regionconsisting of amino acid sequence of SEQ ID NO: 3, and/or a nucleic acidmolecule for coding a heavy chain variable region consisting of aminoacid sequence of SEQ ID NO: 4, but is not limited thereto.

In the vector of the present invention, the nucleic acid molecule forcoding a light chain variable region and the nucleic acid molecule forcoding a heavy chain variable region may be operatively linked to apromoter.

As used herein, the term “operatively linked” means a functional linkagebetween a nucleic acid expression control sequence (e.g., a promoter, asignal sequence or an array of transcriptional regulatory factor bindingsites) and another nucleic acid sequence, wherein the control sequenceregulates the transcription and/or translation of the another nucleicacid sequence.

A recombinant vector system of the present invention may be constructedby means of various methods known in the art, wherein a detailed methodthereof is disclosed in Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press (2001) and this document isincorporated herein by reference.

The vector of the present invention may be typically constructed as avector for cloning or a vector for expression. The recombinant vector ofthe present invention is preferably an expression vector. Also, thevector of the present invention may be constructed in such a way that aprokaryotic cell or a eukaryotic cell is a host.

For example, if the vector of the present invention is the expressionvector and the prokaryotic cell is the host, it is common to include astrong promoter capable of performing transcription (e.g., a tacpromoter, a lac promoter, a lacUV5 promoter, a 1pp promoter, a pLλpromoter, a pRλ promoter, a rac5 promoter, an amp promoter, a recApromoter, an SP6 promoter, a trp promoter, a T7 promoter and the like);a ribosome binding site for initiation of decoding; and atranscription/decoding termination sequence. If E. coli (e.g., HB101,BL21, DH5α, etc.) is used as the host cell, a promoter and operatorregion of E. coli tryptophan biosynthetic pathway (Yanofsky, C., J.Bacteriol., (1984) 158:1018-1024) and a left-oriented promoter of phageλ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980)14:399-445) may be used as a regulatory region. If Bacillus is used asthe host cell, a promoter of a toxoprotein gene of Bacillusthuringiensis (Appl. Environ. Microbiol. (1998) 64:3932-3938; Mol. Gen.Genet. (1996) 250:734-741) or any promoters expressible in Bacillus maybe used as the regulatory region.

Meanwhile, the recombinant vector of the present invention may beprepared by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177,ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1,pHV14, pGEX series, pET series, pUC19 and the like), phages (e.g.,λgt4·λB, λ-Charon, λΔz1, M13 and the like) or virus (e.g., SV40, etc.),which are often used in the art. For example, the recombinant vector ofthe present invention may be prepared by manipulating a pCL expressionvector, particularly a pCLS05 (Korean Patent Registration No.10-1420274) expression vector, but is not limited thereto.

Meanwhile, if the vector of the present invention is the expressionvector and the eukaryotic cell is the host, the followings may be used:the promoters derived from genome of mammal cells (e.g., ametallothionein promoter, a β-actin promoter, a human hemoglobinpromoter and a human muscle creatine promoter) or the promoters derivedfrom mammal virus (e.g., an adenovirus late promoter, a vaccinia virus7.5K promoter, an SV40 promoter, a cytomegalo virus (CMV) promoter, a tkpromoter of HSV, a mouse breast tumor virus (MMTV) promoter, an LTRpromoter of HIV, a promoter of moloney virus, a promoter of epstein-barrvirus (EBV) and a promoter of rous sarcoma virus (RSV)), while generallyhaving a polyadenylation sequence as a transcription terminationsequence. Particularly, the recombinant vector of the present inventionmay contain the CMV promoter.

The recombinant vector of the present invention may be fused with othersequences such that an antibody expressed therefrom may be easilypurified. As a fused sequence, there are, for example, a glutathioneS-transferase (Pharmacia, USA), a maltose binding protein (NEB, USA),FLAG (IBI, USA), 6× His (hexahistidine; Quiagen, USA) and the like.Also, because the protein expressed by means of the vector of thepresent invention is an antibody, such expressed antibody may be easilypurified via a protein A column, etc., without an additional sequencefor purification.

Meanwhile, the recombinant vector of the present invention contains anantibiotic resistance gene conventionally used as a selectable marker inthe art, and may contain, for example, genes resistant to ampicillin,gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin,geneticin, neomycin and tetracycline.

The vector for expressing an antibody according to the present inventionmay be a vector system, in which a light chain and a heavy chain aresimultaneously expressed in one vector, or a system for expressing alight chain and a heavy chain in a separate vector, respectively,wherein both systems are available. In case of the latter one, bothvectors are transferred into a host cell through co-transformation andtargeted transformation. Co-transformation is a method, in which each ofvector DNAs for coding a light chain and a heavy chain is concurrentlytransferred into the host cell, after which a cell of expressing boththe light chain and the heavy chain is selected. Targeted transformationis a method, in which a cell transformed with the vector containing thelight chain (or the heavy chain) is selected, after which a resultingselected cell of expressing the light chain is transformed again withthe vector containing the heavy chain (or the light chain), such that acell of expressing both the light chain and the heavy chain is finallyselected.

2-3. Transformant Containing Recombinant Vector

In another aspect of the present invention, there is provided a hostcell containing the recombinant vector. Preferably, the host cell of thepresent invention is the host cell transformed with the recombinantvector.

The host cell capable of stably and continuously cloning and expressingthe vector of the present invention may be any host cells known in theart, wherein such host cell includes, for example, Bacillus speciesstrains such as Escherichia coli, Bacillus subtilis and Bacillusthuringiensis; and prokaryotic host cells such as Streptomyces,Pseudomonas (for example, Pseudomonas putida), Proteus mirabilis orStaphylococcus (for example, Staphylococcus carnosus), but is notlimited thereto.

A suitable eukaryotic host cell transformed with the vector may be fungisuch as Aspergillus species; yeasts such as Pichia pastoris,Saccharomyces cerevisiae, Schizosaccharomyces and Neurospora crassa;other lower eukaryotic cells; higher eukaryotic cells such asinsect-derived cells; and plant or mammal-derived cells, but is notlimited thereto.

Particularly, the host cell may be monkey kidney cells 7 (COS7), NSOcells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamsterkidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells or 293 cells,and more particularly may be the CHO cells, but is not limited thereto.

In case of using microorganisms such as E. coli, etc., productivitytends to be higher compared to that of using animal cells, etc. However,such use is not preferable to produce intact Ig type antibodies due to aglycosylation problem, but may be used in production of Fab, Fv and thelike.

In the present invention, “transformation” and/or “transfection” intohost cells includes any methods for transferring a nucleic acid intoorganisms, cells, tissues or organs, and may be performed by selecting asuitable standard technology according to host cells as known in theart. The methods as above include electroporation, protoplast fusion,calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl₂)precipitation, agitation with silicon carbide fibers,agrobacteria-mediated transformation, PEG, dextran sulfate,lipofectamine, drying/inhibition-mediated transformation methods and thelike, but are not limited thereto.

2-4. Method for Preparing EMAP II Antibody or Antigen-Binding FragmentThereof

In another aspect of the present invention, there is provided a methodfor preparing an anti-EMAP II antibody or an antigen-binding fragmentthereof, including a step of culturing a transformed cell of the presentinvention. Preferably, the method for preparing an anti-EMAP II antibodyor an antigen-binding fragment thereof according to the presentinvention may further include a step of expressing an EMAP II antibodyor an antigen-binding fragment thereof in cultured transformed cells.

In the method for preparing an antibody or an antigen-binding fragmentthereof, a culture of transformed cells may be performed according toappropriate media and culture conditions known in the art. Such cultureprocess may be easily adjusted and used by those skilled in the artaccording to selected strains. Such various culture methods aredisclosed in various documents (for example, James M. Lee, BiochemicalEngineering, Prentice-Hall International Editions, 138-176). Cellculture is classified into suspension culture and attachment cultureaccording to cell growth modes; and into the batch, fed-batch andcontinuous types of culture methods according to culture methods. Amedium used in culture should appropriately satisfy the requirements forcertain strains.

In animal cell culture, the medium contains a variety of carbon sources,nitrogen sources and microelement components. An example of carbonsources available may include carbohydrates such as glucose, sucrose,lactose, fructose, maltose, starch and cellulose; fats such as soybeanoil, sunflower oil, castor oil and coconut oil; fatty acids such aspalmitic acid, stearic acid and linoleic acid; alcohols such as glyceroland ethanol; and organic acids such as acetic acid, wherein such carbonsources may be used alone or in combination.

A nitrogen source available in the present invention may include, forexample, organic nitrogen sources such as peptone, yeast extract, gravy,malt extract, corn steep liquor (CSL), soybean and wheat; and inorganicnitrogen sources such as urea, ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, whereinsuch nitrogen sources may be used alone or in combination. The mediummay include potassium dihydrogen phosphate, dipotassium hydrogenphosphate and sodium-containing salts corresponding thereto as aphosphorus source. Also, the medium may include metallic salts such asmagnesium sulfate or iron sulfate. Besides, amino acids, vitamins,appropriate precursors and the like may be included therein.

During culture, compounds such as ammonium hydroxide, potassiumhydroxide, ammonia, phosphoric acid and sulfuric acid may be added intoa culture product in an appropriate manner, such that pH of such cultureproduct may be adjusted. During culture, antifoaming agents such asfatty acid polyglycol ester may be also used to inhibit foams from beingcreated. Furthermore, oxygen or oxygen-containing gases (e.g., air) areinjected into the culture product such that the culture product maymaintain an aerobic state thereof. A temperature of the culture productmay be generally 20 to 45° C., preferably 25 to 40° C., but is notlimited thereto.

An antibody obtained from culturing a transformed host cell may be usedin a non-purified state, and may be also used in such a way that theantibody is purified with high purity by further using variousconventional methods, for example, dialysis, salt precipitation,chromatography and the like. Out of them, a method using chromatographyhas been most often used, wherein a type and order of columns may beselected from ion exchange chromatography, size exclusionchromatography, affinity chromatography, etc. according to antibodycharacteristics, culture methods, etc.

3. Use of Anti-EMAP II Antibody or Antigen-Binding Fragment Thereof forDetecting EMAP II Antigen

In another aspect of the present invention, there are provided acomposition for detecting an EMAP II antigen, containing the antibody orthe antigen-binding fragment thereof; a kit for detection containing thesame; and a method for detecting an EMAP II antigen using thecomposition and the kit.

The composition for detecting EMAP II and the kit containing the samemay effectively detect EMAP II, in such a way that the anti-EMAP IIantibody or the antigen-binding fragment thereof according to thepresent invention is brought into contact with a sample to form anantigen-antibody complex.

As used herein, the term “antigen-antibody complex” means a combinationbetween EMAP II and an antibody recognizing the same, so as to identifya presence or abundance of EMAP II in the sample.

A method for quantifying an EMAP II antigen using the composition fordetecting EMAP II and the kit containing the same may be performed byidentifying a formation of an antigen-antibody complex, wherein theformation of the antigen-antibody complex may be identified by means ofenzyme-linked immunosorbent assay (ELISA), western blotting,immunofluorescence, immunohistochemistry staining, flow cytometry,immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay,immunodiffusion assay, complement fixation assay, protein chip or thelike, but is not limited thereto. ELISA includes various ELISA methodssuch as a direct ELISA, which uses a labeled antibody of recognizing anantigen attached to a solid support; an indirect ELISA, which uses alabeled secondary antibody of recognizing a capture antibody in anantibody complex of recognizing an antigen attached to a solid support;a direct sandwich ELISA, which uses another labeled antibody ofrecognizing an antigen in an antibody-antigen complex attached to asolid support; an indirect sandwich ELISA, which carries out reactionwith another antibody of recognizing an antigen in an antibody-antigencomplex attached to a solid support, and then uses a labeled secondaryantibody of recognizing this antibody; etc.

As a label for allowing the formation of antigen-antibody complex to bequalitatively or quantitatively measurable, there are enzymes,fluorescent materials, ligands, luminous materials, microparticles,redox molecules, radio isotopes and the like, but not necessarilylimited thereto. As the enzymes, there are β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkalinephosphatase, acetylcholinesterase, glucose oxidase, hexokinase andGDPase, RNase, glucose oxidase and luciferase, phosphofructokinase,phosphoenolpyruvate carboxylase, aspartate aminotransferase,phosphoenolpyruvate decarboxylase, β-lactamase, etc., but not limitedthereto.

4. Pharmaceutical Use of Anti-EMAP II Antibody or Antigen-BindingFragment Thereof

An antibody or an antigen-binding fragment thereof according to thepresent invention may bind to EMAP II with a high affinity and thus suchantibody may be used in diagnosing, treating or preventing EMAPII-mediated diseases, for example, TNF-α-mediated disease or Alzheimer'sdisease for itself or along with conventional, pharmaceuticallyacceptable carriers. The antibody or the antigen-binding fragmentthereof is the same as described above.

The antibody or the antigen-binding fragment thereof according to thepresent invention may be used in a form of pharmaceutical compositions,quasi drug compositions and health food compositions.

In an aspect of the present invention, there is provided apharmaceutical composition for preventing or treating EMAP II-mediateddiseases, containing the inventive antibody or the antigen-bindingfragment thereof as an active ingredient.

The antibody or the antigen-binding fragment thereof according to thepresent invention effectively inhibits TNF-α secretion by binding toEMAP II and inhibiting an activity thereof, and thus may be used inpreventing or treating TNF-α-mediated diseases.

Particularly, in an aspect of the present invention, there is provided apharmaceutical composition for preventing or treating TNF-α-mediateddiseases, containing the inventive antibody or the antigen-bindingfragment thereof as an active ingredient. The TNF-α-mediated diseasesinclude adult respiratory distress syndrome, lack of appetite, cancer,chronic fatigue syndrome, graft-versus-host rejection, hyperalgesia,inflammatory bowel disease, neuroinflammatory disease, ischemiaincluding cerebral ischemia or reperfusion injury, trauma, epilepsy,brain damage resulting from bleeding or seizure, diabetes, multiplesclerosis, eye disease, pain, pancreatitis, pulmonary fibrosis,rheumatoid arthritis, osteoarthritis, seronegative polyarthritis,ankylosing spondylitis, Reiter's syndrome, reactive arthritis, psoriaticarthritis, enteropathic arthritis, polymyositis, dermatomyositis,scleroderma, systemic sclerosis, vasculitis, cerebral vasculitis,Sjogren's syndrome, rheumatic fever, polychondritis and polymyalgia,rheumatoid and giant cell arteritis, septic shock, side effectsresulting from radiotherapy, systemic lupus erythematosus,temporomandibular disease and thyroiditis, but are not limited thereto.

In another aspect of the present invention, there is provided apharmaceutical composition for preventing or treating Alzheimer'sdisease, containing the inventive antibody or the antigen-bindingfragment thereof as an active ingredient.

As used herein, the term “prevention” means all the acts, which inhibita disease or delay a progress thereof by administering the compositionof the present invention, while “treatment” means the inhibition,reduction or elimination of a development of a disease.

A pharmaceutical composition of the present invention may furthercontain pharmaceutically acceptable carriers, wherein thepharmaceutically acceptable carriers are ones conventionally used informulating a preparation, including, but not limited thereto, lactose,dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calciumphosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc,magnesium stearate, mineral oil and the like. A composition forpreventing or treating cancer according to the present invention mayfurther contain lubricants, humectants, sweetening agents, flavoringagents, emulsifiers, suspending agents, preservatives, etc. in additionto the components above. Suitable, pharmaceutically acceptable carriersand preparations are described in detail in a document [Remington'sPharmaceutical Sciences (19th ed., 1995)].

The pharmaceutical composition of the present invention may be orally orparenterally administered, wherein the parenteral administration may beperformed by means of intravenous infusion, subcutaneous infusion,intramuscular infusion, intraperitoneal infusion, endodermisadministration, local administration, intranasal administration,intrapulmonary administration, intrarectal administration and the like.Upon oral administration, protein or peptide is digested. Thus, an oralcomposition may be formulated into dosage forms in such a way that anactive drug thereof is coated or protected from being decomposed in thestomach, wherein the composition of the present invention may beadministered by means of any devices capable of moving an activesubstance into a target cell.

A suitable dosage of the pharmaceutical composition according to thepresent invention may vary depending on factors such as a method forformulating a preparation, an administration mode, a patient's age,weight, gender, pathological condition, food, administration time,administration route, excretion rate and reaction sensitivity, whereinordinarily skilled doctors may easily determine and prescribe aneffective dosage for a desired treatment or prevention. According to oneexemplary embodiment of the present invention, a daily dosage of theinventive pharmaceutical composition may be 0.1-100 mg/kg, preferably0.1-10 mg/kg and more preferably 0.1-2 mg/kg. As used herein, the term“pharmaceutically effective amount” means an amount enough to treat,prevent and diagnose diseases.

The pharmaceutical composition of the present invention may beformulated into preparations by using pharmaceutically acceptablecarriers and/or excipients according to a method easily practicable bythose skilled in the art to which the present invention pertains, andthus may be prepared in a unit dose form or prepared by being insertedinto a multi-dose container. At that time, a dosage form may be in aform of solutions, suspensions or emulsions in oily or aqueous media, ormay be in a form of extracts, powders, suppositories, granules, tabletsor capsules, and may further contain dispersing agents or stabilizingagents.

The composition of the present invention may be administered as anindividual therapeutic agent or in combination with other therapeuticagents, and may be administered sequentially or simultaneously with aconventional therapeutic agent.

An antibody or an antigen-binding fragment thereof according to thepresent invention may be administered in vivo in a form ofantibody-drugs (functional molecules) and dual specificity antibody-drug(functional molecules) conjugates and thus may be used in treatingcancer, wherein descriptions thereof are the same as described above.Several appropriate and preferred conditions for targeting drugs intospecific target regions are reported, for example, in a document [Trouetet al., Plenum Press, New York and London, (1982) 19-30].

The antibody or the antigen-binding fragment thereof according to thepresent invention may be further combined with functional molecules oradministered in combination therewith, and thus may be used inpreventing, treating and diagnosing EMAP II-related diseases. Thefunctional molecules may include chemicals, radioactive nuclides,immunotherapy agents, cytokine, chemokine, toxin, biological agents,enzyme inhibitors and the like.

In one specific embodiment of the present invention, it was identifiedin case of administering the pharmaceutical composition of the presentinvention into a collagen-induced arthritis (CIA) mouse model that thereis distinctively an alleviation in arthritis scores and a decrease infoot thickness (FIG. 4), and that there is an excellent effect ofinhibiting TNF-α levels in blood (FIG. 5). Thus, the pharmaceuticalcomposition of the present invention may be used as a therapeutic agentfor EAMP II-mediated diseases, including TNF-α-mediated disease.

In another aspect of the present invention, there is provided a methodfor diagnosing, preventing or treating EMAP II-mediated diseases, forexample, TNF-α-mediated disease or Alzheimer's disease, including a stepof administering a pharmaceutically effective amount of the inventiveantibody or the antigen-binding fragment thereof into subjects, e.g.,humans or mammals excluding the humans. Also, the present inventionprovides a method for diagnosing EMAP II-mediated diseases, for example,TNF-α-mediated disease or Alzheimer's disease, including a step ofadministering the inventive antibody or the antigen-binding fragmentthereof into subjects in need, e.g., humans or mammals excluding thehumans.

As used herein, the term “individual” means all the animals includingmonkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats,dogs, mice, rats, rabbits or guinea pigs, as well as humans, who havedeveloped the EMAP II-mediated diseases or are likely to do so.

In another aspect of the present invention, there is provided a use ofthe inventive antibody or the antigen-binding fragment thereof inpreparing a drug for preventing or treating EMAP II-mediated diseases,for example, TNF-α-mediated disease or Alzheimer's disease.

Advantageous Effects

An anti-EMAP II antibody of the present invention binds to an EMAP IIantigen with a higher affinity and exhibits a more excellent effect ofpreventing and treating EMAP II-mediated diseases, for example,TNF-α-mediated disease or Alzheimer's disease compared to existingantibodies.

Thus, the antibody or the antigen-binding fragment thereof according tothe present invention maybe valuably used in detecting an EMAP IIantigen, or preventing, treating or diagnosing EMAP II-mediateddiseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of identifying an anti-EMAP II antibody of thepresent invention under reducing conditions by using SDS-PAGE.

FIG. 2 shows results of comparing the antigen-binding abilities of theinventive anti-EMAP II antibody by using ELISA.

FIG. 3 shows results of comparing the antigen-binding abilities of theinventive anti-EMAP II antibody by using SPR assay.

FIG. 4 shows an effect of the inventive anti-EMAP II antibody oninhibiting arthritis in a CIA mouse model.

FIG. 5 shows an efficacy of the inventive anti-EMAP II antibody oninhibiting TNF-α secretion in the CIA mouse model.

MODE FOR INVENTION

Hereinafter, the present invention will be described in more detailthrough exemplary embodiments. These exemplary embodiments are providedonly for the purpose of illustrating the present invention in moredetail, and thus it will be apparent to those skilled in the art thatthe scope of the present invention is not limited thereto according tothe gist of the present invention.

Example 1. Preparation for Anti-EMAP II Antibody Example 1-1. SecuringAmino Acid Sequence for Anti-EMAP II Antibody Variant Variable Region

Affinity maturation was performed to induce a mutation from light chainand heavy chain variable regions of BAP015-hum03 antibody (commissionedto Bioatla LLC), for the purpose of obtaining an anti-EMAP II antibodyvariant having a higher antigen affinity, a higher effect of inhibitingTNF-α secretion and a higher effect of treating EMAP II-mediateddiseases compared to a known human anti-EMAP II antibody (BAP015-hum03)(Korean Patent Publication No. 2012-0118918).

A sequence of the BAP015-hum03 antibody variant obtained above wasanalyzed to identify an amino acid sequence of a complementarydetermining region (CDR) of an antibody variable region for each ofantibody variants (Table 1).

TABLE 1 Classifi- Amino acid No. cation sequence SEQ ID NO:  1 L-CDR1KASQDVSTAVA SEQ ID NO: 7  2 L-CDR2a SASYRYT SEQ ID NO: 8  3 L-CDR2bSASYRYW SEQ ID NO: 9  4 L-CDR2c SASYGYT SEQ ID NO: 10  5 L-CDR3QQHYSIPYT SEQ ID NO: 11  6 H-CDR1 GYTFTSYTMH SEQ ID NO: 12  7 H-CDR2aYINPSSGFTNYNQKFKD SEQ ID NO: 13  8 H-CDR2b YINPRSGFTNYRQKFKHSEQ ID NO: 14  9 H-CDR2c YTNPSSGFTNYTQKFKD SEQ ID NO: 15 10 H-CDR3 RFAYSEQ ID NO: 16

The light chain CDR1, CDR2 and CDR3 regions of BAP015-hum03 antibodyconsist of L-CDR1, L-CDR2a and L-CDR3 amino acid sequences,respectively, and the heavy chain CDR1, CDR2 and CDR3 regions thereofconsist of H-CDR1, H-CDR2a and H-CDR3 amino acid sequences,respectively. As a variable region of BAP015-hum03 antibody variant, alight chain variable region containing L-CDR1 (SEQ ID NO: 7), L-CDR2b(SEQ ID NO: 9) and L-CDR3 (SEQ ID NO: 11) was named BAP072-LC-T056W; anda light chain variable region containing L-CDR1 (SEQ ID NO: 7), L-CDR2c(SEQ ID NO: 10) and L-CDR3 (SEQ ID NO: 11) was named BAP072-LC-R54G,respectively, wherein a heavy chain variable region containing H-CDR1(SEQ ID NO: 12), H-CDR2b (SEQ ID NO: 14) and H-CDR3 (SEQ ID NO: 16) wasnamed BAP072-CPS-15-HC; and a heavy chain variable region containingH-CDR1 (SEQ ID NO: 12), H-CDR2c (SEQ ID NO: 15) and H-CDR3 (SEQ ID NO:16) was named BAP072-CPS-08-HC, respectively.

With regard to light chain and heavy chain variable regions of theBAP015-hum03 antibody and the BAP015-hum03 antibody variant preparedabove, the entire amino acid sequences thereof are as follows (Table 2).

TABLE 2 Peptide CDR name constitution Amino acid sequence Mutation LightBAP015- L-CDR1 AIQLTQSPSS LSASVGDRVT Reference chain hum03-LC L-CDR2aITCKASQDVS sequence variable (SEQ IN L-CDR3 TAVAWYQQKP region NO: 1)GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTFTISRLEP EDFAVYYCQQ HYSIPYTFGQ GBAP072-LC- L-CDR1 AIQLTQSPSS LSASVGDRVT T056W T056W L-CDR2b ITCKASQDVS(SEQ ID L-CDR3 TAVAWYQQKP NO: 3) GKAPKLLIYS ASYRYWGVPSRFSGSGSGTD FTFTISRLEP EDFAVYYCQQ HYSIPYTFGQ G BAP072-LC- L-CDR1AIQLTQSPSS LSASVGDRVT R054G R54G (SEQ L-CDR2c ITCKASQDVS ID NO: 5)L-CDR3 TAVAWYQQKP GKAPKLLIYS ASYGYTGVPS RFSGSGSGTD FTFTISRLEP EDFAVYYCQQHYSIPYTFGQ G Heavy BAP015- H-CDR1 EVQLVQSGAE Reference chain hum03-HCH-CDR2a VKKPGATVKI SCKVSGYTFT sequence variable (SEQ ID H-CDR3SYTMHWVRQA region NO: 2) PGQGLEWMGY INPSSGFTNY NQKFKDRVTI SADKSISTAYLQWSSLKASD TAMYYCASRF AYWGQG BAP072- H-CDR1 EVQLVQSGAE S054R, CPS-15-HCH-CDR2b VKKPGATVKI SCKVSGYTFT N061R, (SEQ ID H-CDR3 SYTMHWVRQA D066H,NO: 4) PGQGLEWMGY A097F INPRSGFTNY RQKFKHRVTI SADKSISTAY LQWSSLKASDTAMYYCFSRF AYWGQG BAP072- H-CDR1 EVQLVQSGAE I051T, CPS-08-HC H-CDR2cVKKPGATVKI SCKVSGYTFT N061T, (SEQ ID H-CDR3 SYTMHWVRQA T069H, NO: 6)PGQGLEWMGY M093W TNPSSGFTNY TQKFKDRVHI SADKSISTAY LQWSSLKASDTAWYYCASRF AYWGQG

As can be seen in the table above, it was identified for theBAP015-hum03 antibody variant obtained from affinity maturation thatboth a light chain and a heavy chain thereof hold the identical CDR1 andCDR3 to a parent antibody and both the light chain and the heavy chainthereof have one to three amino acid substitution mutations developed ata sequence of CDR2.

Example 1-2. Preparation for Expression Vector of Anti-EMAP II AntibodyVariant Gene

A light chain variable region (SEQ ID NO: 1) and a heavy chain variableregion (SEQ ID NO: 2) of an anti-EMAP II parent antibody, as well as alight chain variable region (SEQ ID NO: 3 or SEQ ID NO: 5) and a heavychain variable region (SEQ ID NO: 4 or SEQ ID NO: 6) of an antibodyvariant obtained in Example 1-1, were cloned into each of the mammalexpression vector pBAK2b (BioAtla LLC) systems, respectively.

In the vector, each light chain variable region was fused with a framefor a human kappa constant region, and a heavy chain variable region wasfused with a frame for a human IgG1 constant region. Also, in thevector, a leader peptide sequence for secretion of a full-length IgG1antibody in a medium was added into a gene. A gene cloning method is thesame as known to those skilled in the art.

In result, each of antibodies BAP072-EMAP II-1, BAP072-EMAP II-2,BAP072-EMAP II-3 and BAP072-EMAP II-4 was produced in such a way that alight chain variable region and a heavy chain variable region werecombined respectively (Table ₃).

TABLE 3 Light chain Heavy chain Constant Antibody name variable regionvariable region region BAP₀₁₅-hum₀₃ BAP₀₁₅- BAP₀₁₅-hum₀₃- Human kappahum₀₃-LC HC light chain BAP₀₇₂-EMAP II-₁ BAP₀₇₂-LC- BAP₀₇₂-CPS-₁₅-constant region T₀₅₆W HC and Human BAP₀₇₂-EMAP 11-₂ BAP₀₇₂-LC-BAP₀₇₂-CPS-08- IgG₁ heavy T₀₅₆W HC chain constant BAP₀₇₂-EMAP 11-₃BAP₀₇₂- BAP₀₇₂-CPS-₁₅- region LC-R₅₄G HC BAP₀₇₂-EMAP 11-₄ BAP₀₇₂-BAP₀₇₂-CPS-08- LC-R₅₄G HC

Five clones of each vector were subjected to sequencing to identify anincorporation and sequence of LC and HC reading frames within anexpression vector, after which three clones were selected for anexpression test within CHO cells. After that, a glycerol stock of thethree clones was prepared, after which a plasmid DNA without endotoxinwas prepared for the expression test within the CHO cells.

Example 1-3. Detection of Anti-EMAP II Antibody Variant afterTransformation into CHO Cells

With regard to CHO-S cells, transformation was performed by using theplasmid DNA obtained above.

First of all, the CHO-S cells (CD-CHO) (Invitrogen) were transferredinto a single-layer culture product within serum-supplement D-MEM(Dulbecco's Modified Eagle Medium) (Invitrogen) one week beforetransfection. 0.4×10⁵ cells were plated into 100

of the serum-supplement D-MEM per transfection sample in a 96-wellformat one day before transfection. A DNA-lipofectamine complex wasprepared for each transfection sample. 0.2 μg of plasmid DNA was dilutedin 25

of Opti-MEM reduced serum medium and mixed therein. 0.5

of lipofectamine 2000 (Invitrogen) was diluted in 25

of Opti-MEM reduced serum medium. The resulting one was mixed andincubated for 5 minutes at room temperature. The diluted DNA andlipofectamine were mixed together and incubated for 20 minutes at roomtemperature. 50

of the DNA-lipofectamine complex was added into each well containing thecells and the medium and softly mixed together. The resulting cells wereincubated overnight under the condition of 5% CO₂ and 37° C. The mediumwas removed from each well by suction, after which 100

of serum-supplement D-MEM was added into each well.

In 48 hours after transfection, a cell culture supernatant was collectedtherefrom, after which ELISA was performed to quantify a concentrationof recombinant IgG in such collected cell culture supernatant. First ofall, IgG of recombinant BAP015-hum03, BAP072-EMAP II-1, BAP072-EMAPII-2, BAP072-EMAP II-3 and BAP072-EMAP II-4 was captured in the cellculture supernatant by using an anti-human Fc antibody fixed to theplate. A bound recombinant IgG was detected with an anti-human IgG HRPconjugate, and quantified by using a commercial human IgG as a referencestandard.

More particularly, a Nunc-Immuno Maxisorp 96-well plate (Nalge Nunc)containing 100

of affinity-purified Fc specific goat anti-human IgG (Sigma) at 10 μg/

was coated in a coating solution, after which the plate was sealed upand incubated overnight at 4° C. After that, the plate was washed with200

of washing solution by stirring at 200 rpm for 5 minutes at roomtemperature. Then, 200

of blocking solution was added thereinto and stirred at 200 rpm for 1hour at room temperature. A purified human serum IgG (Invitrogen) at astandard concentration of 100 μg/

or 100

of the supernatant obtained from transfection was redundantly addedthereinto. The resulting one was stirred at 200 rpm for 1 hour at roomtemperature, and washed twice with 200

of washing solution at 200 rpm for 5 minutes at room temperature. 100

of 1:5000 dilution of HRP-conjugated affinity-purified goat anti-humanantibodies (Promega) in blocking solution was added into each well, thenstirred at 200 rpm for one hour at room temperature, and then washedthree times with 200

of washing solution at 200 rpm for five minutes at room temperature.Sigma TMB substrate was added into each well and incubated at roomtemperature. After that, 100

was added thereinto to finish a reaction and read at 450 nm.

Example 1-4. Isolation and Purification of Anti-EMAP II Antibody Variant

The CHO-S cell lines with each DNA transformed therein were cultured asdescribed above, after which culture fluid was collected therefrom andprotein A agarose bead (Invitrogen) was loaded thereon, such that eachantibody was allowed to bind thereto. Then, the bead was washed withPBS, then eluted with 0.1M glycine (pH 3.0), then neutralized with 1MTris-HCl (pH 8.0), and then dialyzed again with PBS containing 20%glycerol, such that antibodies (BAP015-hum03, BAP072-EMAP II-1,BAP072-EMAP II-2, BAP072-EMAP II-3 and BAP072-EMAP II-4) were identifiedwith SDS-PAGE gel under the non-reducing conditions and under thereducing conditions, respectively (FIG. 1). The antibodies obtained werekept at 70° C.

Example 2. Identification of Antigen-Binding Ability of Anti-EMAP IIAntibody Variant Example 2-1. Comparison of Antigen-Binding Abilities ofAntibody Variants by ELISA

A matrix experiment was set up as follows, in order to identify a linearscope for comparing the antigen-binding abilities of BAP015-hum03,BAP072-EMAP II-1, BAP072-EMAP II-2, BAP072-EMAP II-3 and BAP072-EMAPII-4 antibodies.

Each well of a 96-well plate was coated with 100

of EMAP II antigens at a concentration of 1 μg/ml, and incubated at 37°C. for 2 hours. The coated antigens were blocked with 5% BSA and washedwith washing buffer solution. Then, the antigens were treated with adilution of BAP015-hum03, BAP072-EMAP II-1, BAP072-EMAP II-2,BAP072-EMAP II-3 and BAP072-EMAP II-4 antibodies (at 0.625, 1.25, 2.5,5, 10, 20, 50, 100, 500 and 1000 μg/ml), and incubated for 2 hours. Theresulting ones were washed 3 times with washing buffer solution, thentreated with anti-IgG-HRP antibodies, and then incubated for 1 hour.Then, the resulting ones were washed 3 times with washing buffersolution, and treated with a substrate for 30 minutes at roomtemperature. Color development was performed, after which stop solutionwas inserted thereinto, such that absorbance was measured at awavelength of 450 nm to test an antigen-binding ability of theantibodies.

In result, in case of BAP072-EMAP II-2, BAP072-EMAP II-3 and BAP072-EMAPII-4 antibodies, a concentration of antibodies to reach O.D 2.5 is about100 ng/ml or more, thus showing a level of binding ability equal toBAP015-hum03 antibody. However, in case of BAP072-EMAP II-1 antibody,the concentration of antibodies to reach O.D 2.5 is no more than about10 ng/ml, thus showing that an antigen-binding ability thereof is about10 times more than other antibodies (FIG. 2).

Example 2-2. Comparison of Antigen-Binding Abilities of Anti-EMAP IIAntibody Variants Through SPR Analysis

A surface plasmon resonance (SPR) method was used to further compare theantigen-binding abilities between BAP015-hum03 and BAP072-EMAP II-1.Reichert SR7500DC system (Reichert Technologies, Depew, N.Y.) was usedfor SPR, and Scrubber2 software was used for data collection. 2.5 μg ofEMAP II protein was fixed to PEG chip (Reichert Technologies), afterwhich BAP015-hum03 and BAP072-EMAP II-1 antibodies were flowed thereintoto compare the binding abilities therebetween.

In result, a K_(D) value of BAP015-hum03 antibody is 7.09×10⁻¹⁰M and theK_(D) value of BAP072-EMAP II-1 antibody is 6.3×10⁻¹¹M, thus identifyingthat BAP072-EMAP II-1 shows an antigen-binding ability about 10 timesmore than a control group, i.e., BAP015-hum03, just as shown in theELISA results above (FIG. 3).

Example 3. Therapeutic Effect on Mouse Arthritis

A collagen-induced arthritis (CIA) mouse model was used to identify aneffect of the inventive anti-EMAP II antibody on treating arthritis. Toprepare a model mouse, six-week-old pathogen free DBA/1 mice (female)were purchased from Orient Bio and kept until they became seven weeksold.

For primary immunization, 200 mg of native bovine type II collagen(Chondrex) was dissolved in 100 ml of 10 mM acetic acid and fully mixedby stirring at 1000 rpm for 12 hours at 4° C. with the same amount ofcomplete Freund's adjuvant containing 200 mg of inactivatedmycobacterium tuberculosis (Sigma-Aldrich). 100 ml of a resultingmixture was intradermally injected into a basal region in each tail ofseven-week-old female DBA/1 mice to perform primary immunization.

On 21st day after the primary immunization, the mice were boosted with100 mg of bovine type II collagen in incomplete Freund's adjuvant(Sigma-Aldrich) to perform secondary immunization.

For 70 days after the primary immunization, the mice were observed oncea week to evaluate the severity of their arthritis. A thickness of eachmouse foot was measured with a vernier caliper. Two independentobservers evaluated a total of four legs of each mouse and gavearthritis scores ranging from 0 to 5 (Table 4).

TABLE 4 Arthritis scores Symptoms 0 No sign of arthritis 1 One rednessin feet or toes 2 Slight swelling in an ankle with a few individual toesswollen 3 Moderate swelling in an ankle 4 Severe enlargement of anentire ankle including toes 5 Inflammable limbs involving multiplejoints

In 14 days after secondary immunization, it was observed that CIA micewere completely developed. 50 mice, which had an arthritis score of morethan 4 points, were equally divided into six groups (Group 1: normalcontrol; Group 2: treated with vehicle; Group 3: treated with 5 mg/kg ofBAP015-hum03; Group 4: treated with 1 mg/kg of BAP072-EMAP II-1; Group5: treated with 2 mg/kg of BAP072-EMAP II-1; and Group 6: treated with 5mg/kg of BAP072-EMAP II-1). Then, arthritis scores were measured for allthe mice but the normal control group, after administering anintraperitoneal injection with the antibodies at a concentration markedin the groups a total of 4 times at an interval of once a week.Arthritis scores were measured in a double-blind manner.

In result, it was identified that an arthritis symptom starts to bealleviated from one week after treatment with antibodies. When making acomparison between the groups treated with 5 mg/kg of the antibodies,BAP072-EMAP II-1 antibody started to show an excellent alleviation ofarthritis and a great decrease in the thickness of swollen feet comparedto the control group, i.e., BAP015-hum03 antibody from one week aftertreatment, thus achieving a therapeutic effect early on (FIG. 4).Furthermore, the therapeutic effect of the control group, i.e., 5 mg/kgof BAP015-hum03 antibody was similar to that of 2 mg/kg of BAP072-EMAPII-1 antibody according to the present invention, thus identifying thatthe present invention has an excellent therapeutic efficacy of achievinga level of alleviating arthritis similar to existing antibodies even ina low dose. As identified in Example 2, such results were based on anexcellent antigen-binding capacity of BAP072-EMAP II-1 according to thepresent invention, thus proving that the present invention has anexcellent efficacy of treating arthritis.

Example 4. Inhibitory Effect on Mouse TNF-α Secretion

A TNF-α level in serum of CIA mice treated with antibodies wasinvestigated with ELISA, in order to identify an improved effect of theinventive anti-EMAP II antibody on inhibiting TNF-α secretion. ELISA wasperformed according to a manufacturer's instructions, after purchasing amouse TNF-α ELISA kit (R&D systems, USA). Experiment methods such aspreparation for the CIA mouse and a schedule for administeringantibodies are the same as described in Example 3. Blood was sampledonce a week from the CIA mice prepared in Example 3-1 to isolate serumtherefrom, after which ELISA was performed for TNF-α.

In result, it was shown that both BAP015-hum03 and BAP072-EMAP II-1antibodies tend to show a significant decrease in a TNF-α level, i.e.,an inflammatory factor (FIG. 5). Out of them, when comparing the resultsof TNF-α level between BAP015-hum03 and BAP072-EMAP II-1, it was shownthat BAP072-EMAP II-1 antibody of the present invention decreases theTNF-α level more remarkably than BAP015-hum03 antibody.

In other words, the BAP072-EMAP II-1 antibody of the present inventioneffectively inhibits TNF-α, i.e., a representative inflammation marker,thus showing an excellent effect of treating TNF-α-mediated diseasesincluding arthritis.

The embodiments above show that the EMAP II antibody variant or theantigen-binding site thereof according to the present invention has amuch more antigen-binding capacity as well as a much more excellenteffect on EMAP II-mediated diseases compared to EMAP II antibodiesdisclosed in the prior document. In particular, the EMAP II antibodyvariant of the present invention has an excellent effect of inhibitingTNF-α and thus exhibits a remarkable effect of treating TNF-α-mediateddiseases.

While specific portions of the present invention have been described indetail above, it is apparent to those having ordinary skill in the artthat such detailed descriptions are set forth to illustrate exemplaryembodiments only, but are not construed to limit the scope of thepresent invention. Thus, it should be understood that the substantialscope of the present invention is defined by the accompanying claims andequivalents thereto.

1.-16. (canceled)
 17. A method for treating neuroinflammatory disease orAlzheimer's disease, including administering a therapeutically effectiveamount of a composition comprising an anti-EMAP II antibody or theantigen-binding fragment thereof, wherein the anti-EMAP II antibody orthe antigen-binding fragment thereof, comprising a light chain variableregion, containing a light chain CDR1 consisting of SEQ ID NO: 7, alight chain CDR2 consisting of SEQ ID NO: 9, and a light chain CDR3consisting of SEQ ID NO: 11; and a heavy chain variable region,containing a heavy chain CDR1 consisting of SEQ ID NO: 12, a heavy chainCDR2 consisting of SEQ ID NO: 14, and a heavy chain CDR3 consisting ofSEQ ID NO:
 16. 18. The method according to claim 17, wherein theantibody comprises a light chain variable region consisting of SEQ IDNO: 3; and a heavy chain variable region consisting of SEQ ID NO:
 4. 19.The method according to claim 17, characterized in that the antibody isa humanized antibody.
 20. The method according to claim 17, wherein theantibody comprises a kappa or human IgG-derived constant region.
 21. Themethod according to claim 17, characterized in that the antigen bindingfragment is Fab, F(ab′), F(ab′)2 or Fv.